CPS11, CPS45, and CPS49 thalidomide analogs inhibit NF-κB, activate NFAT, and repress cytokine expression through elevated ROS. (A) Top panel shows immunoblot of phospho-IκBα, IκBα, and actin in the cytosol of Jurkat cells stimulated with PHA/PMA for 1 hour in the presence or absence of 10 μM CPS45. Bottom panel shows immunoblot of phospho-IκBα,IκBα, phospho-RelA, and RelA in whole-cell extracts (WCE) of Jurkat cells preincubated for 15 minutes with 10 μM CPS45 prior to stimulation with PHA/PMA for 15 minutes. (B) CPS45 (10 μM) stimulation of NFAT-mediated transactivation alone or in the presence of PHA/PMA, cyclosporin A, or both combined. Data are an average of triplicate measurements and each is representative of at least 2 independent experiments. Error bars indicate standard error of the mean (SEM). (C) PCA of the dose- and time-dependent influence of thalidomide, CPS11, CPS45, and CPS49 on mitogen-induced secretion of GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, and TNF-α in Jurkat cells (mitogen list is in Figure 1B). (D) Hierarchical clustering comparison of the dose- and time-dependent influence of thalidomide, CPS11, CPS45, and CPS49 on the mitogen-induced cytokine secretion pattern of Jurkat cells. (E) FACS profile of cells loaded with the oxidation-sensitive dye DCFDA and treated with either 10 μM thalidomide, CPS11, CPS45, or CPS49 for 60 minutes. Percentages of maximum cell counts are plotted against fluorescence intensity. (F) FACS profile of the levels of ROS generated after 45 minutes of treatment with 10 μM CPS45 (left panel) compared with cells treated with PHA/PMA alone or PHA/PMA and CPS45 combined (right panel).